Note – our collaborators also call it PoxA
Description: This is an usual lysyl tRNA synthetase, also named Lysine-tRNA ligase, which belongs to the class II aminoacyl-tRNA synthetases. The structure shows an anti-parallel beta-sheet fold flanked by alpha-helices and was found as a dimer, however it lacks an tRNA anticodon-binding domain. For example, comparing it to the E. coli lysyl tRNA synthetase (LysU) residues 5-325 of PoxA overlay with residues 167-502 of LysU (in complex with AMPCP and lysine, PDB code 1E22) with an RMSD of 1.9 A, a Z-score of 11.4 and 45 residues are identical in this alignment. A similar structure apparently has been solved in Pyrococcus furiosis (PDB code 1NNH); this structure also is a smaller version of a tRNA synthetase (294 residues) with a catalytic domain and no anticodon-binding domain; this protein overlays with the PoxA structure with a Z-score of 10.1 and an RMSD of 1.9 A. Crystals of the apo-structure were hard to achieve; however the protein crystallized readily in the presence of ATP. Our first structure contains a single bound AMP for each PoxA molecule; from the structure residues binding the nucleotide include F112 and R303, whose side chains stack on either side of the adenine ring, E102 whose carboxyl group hydrogen-bonds to the N2 NH of the adenine ring, and R100, whose guanidium group forms a salt bridge with the alpha phosphate of the AMP. In overlaying the structure of 1E22 (LysU plus AMPCP) with the PoxA/AMP structure, one observes an additional phosphate group in the PoxA structure occupying a position midway between the beta and gamma phosphates of the AMPCP molecule in the LysU structure. This phosphate is hydrogen-bonded by the side chains of H108 and E244.
At present we are working on solving the structure of PoxA with a non-hydrolyzable ATP analogue. Our collaborator William Navarre at University of Toronto has been involved in determination of its amino acid substrate and its role in Salmonella pathogenesis, and this combination of structural, biochemical and microbiological work will form the basis of a manuscript in the not to near future.